Acute changes in pulmonary blood flow (PBF) are an integral part of surgical repair of many congenital heart defects. In patients with single ventricle anatomy and physiology, the post-operative balance of blood flow to the lungs and body is often dictated by the resistances of the respective vascular beds. In fact, post-operatively the dynamic changes in vascular resistance can lead to significant morbidity and mortality, and much of our therapies are focused on altering pulmonary vascular resistance in attempt to optimize cardiac output while maintaining enough pulmonary flow to ensure adequate systemic oxygenation. In our initial project, we characterized novel chronic alterations and interactions in nitric oxide (NO) and endothelin-1 (ET-1) signaling during a model of increased PBF secondary to congenital heart disease in the lamb. We have recently demonstrated that NO and ET-1 may also acutely co-regulate each other, via a superoxide-dependent mechanism, and that these interactions mediate dynamic changes in vascular tone following acute changes in fetal PBF. Based on our preliminary data, we hypothesize that NO-ET-1 interactions mediate the dynamic changes in pulmonary vascular resistance immediately following surgically induced changes in PBF. In particular, we hypothesize that via ETA receptor signaling, superoxide-mediated decreases in NO activity and increased ET-l-induced vasoconstriction limit increases in PBF following surgery. In this renewal application, we will utilize an integrated physiologic, biochemical, cellular, and molecular approach to investigate this hypothesis and its mechanisms in 3 proposed Aims. In Aim 1, we plan to indirectly investigate the effect of changes in PBF and pressure on NO and ET-1 gene expression and activity in clinically relevant whole animal models, and potential developmental differences. In Aim 2, we will utilize whole animal models, smooth muscle cell cultures, and endothelial/smooth muscle cell co-cultures to investigate the effects of NO/ET-1 interactions in regulating changes in PBF and pressure, the potential role of reactive oxygen species in these interactions, and potential developmental differences. In Aim 3, we will utilize whole animal models, endothelial cell cultures, and co-cultures to determine the independent effects of pressure vs. flow in this process. A better understanding of these mechanisms may improve perioperative treatment strategies and reduce morbidity and mortality.